1. Technical Field
The present invention relates to a more rapid process for reducing certain nitrogen-containing compounds present in tobacco materials. More specifically, the present invention employs a vacuum during the incubation step of a process for dissimilatory reduction of nitrate in tobacco materials, whereby the reduction of nitrate to nitrogen gas is expedited.
2. Description of Prior Art
It is generally recognized that smoking products having lowered amounts of oxides of nitrogen present in smoke are desirable. Therefore, a number of methods have been developed to reduce the delivery of oxides of nitrogen by smoking products. Among these techniques are various methods wherein the nitrate content of the tobacco is altered. For example, methods involving microbial treatment of tobacco to accomplish such nitrate reduction have been proposed.
Specifically in Gaisch et al. Belgian Pat. No. 886,445 published Aug. 14, 1978 and assigned to Fabriques de Tabac Reunies S. A. a process for degrading nitrates and nitrites in tobacco to nitrogen or ammonia compounds by means of microorganisms which would normally require oxygen, but are capable of anaerobic denitration is described. Gaisch et al. German Offenlegungsschrift 28 16427, filed Apr. 15, 1978 and published Nov. 9, 1978, describes a process for microbial degradation of nitrate, nitrite and other nitrogen containing compounds in tobacco. According to Gaisch et al., under nitrogen deficiency or oxygen deficiency conditions, the microorganisms employed obtain their nitrogen or oxygen requirements respectively from nitrate or nitrite degradation. The microorganisms which can be used in these two processes may be selected from the genus Aerobacter, Pseudomonas, Micrococcus of Escherichia, with Enterobacter aerogenes being specifically employed in the examples.
In parent copending application Ser. No. 900,044 filed on Apr. 25, 1978, the applicants herein describe a process for microbial reduction of nitrates in tobacco via a dissimilatory denitrification pathway whereby nitrogen gas is the end product. The microorganism specifically suggested for use in the process is Paracoccus denitrificans or Micrococcus denitrificans. Species of the genera Pseudomonas, Alcaligenes, Bacillus and Propionibacterium can also be employed.
Further U.S. Pat. No. 3,845,774 to Tso et al. describes tobacco treatment methods referred to as homogenized leaf curing wherein the tobacco is homogenized and incubated during curing in order to regulate the composition of the final product. Nitrate-nitrogen and total nitrogen are reduced somewhat; however, the amount of reduction is not as significant as that of the present process. Although Tso et al. allude to the fact that tabacco modification can be accomplished by the use of additional techniques during homogenization and incubation, such as enzyme and microbial action, no specific methods or means for reducing nitrate-nitrogen are suggested.
Gravely et al., U.S. Pat. No. 3,747,608 relates to a method for aerobic microbial digestion of pectin-bound plant material, specifically tobacco materials. Although the invention deals predominantly with methods for fibrilating tobacco materials using pectolytic enzyme-producing microorganisms, Examples 11, 13 and 14 disclose data related to the concomitant denitration of tobacco using the microorganism Erwinia carotovora, ATCC 495. This microorganism is unsuitable for use in the present invention since pectolytic enzyme-producing microorganisms, such as Erwinia carotovora, destroy the structural integrity of the tobacco.
W. O. Atkinson et al. reported a reduction in various tobacco leaf components, including nitrate-nitrogen, by varying homogenization and incubation techniques during curing. (Abstract of Proceedings of the University of Kentucky Tobacco and Health Research Institute, Lexington, Ky., Conference Report 4, March 1973, pages 829-33.)
Denitration by means of microorganisms is also known outside the tobacco arts. Representative examples are U.S. Pat. Nos. 3,709,364 to Savage, 3,829,377 to Hashimoto, 4,039,438 to Anderson, and 4,043,936 to Francis et al. which describe denitrification of waste water using anaerobic bacteria to reduce the nitrate to nitrogen gas. Members of the Thiobacillus, Pseudomonas, Chromobacter, Bacillus and Clostridium genera are among the microorganisms which may be employed. In the Hashimoto patent the use of pressurized systems to increase the amount of methane available to the microorganisms and to facilitate liberation of the nitrogen gas by venting are suggested. The Anderson patent suggests conducting the process at ambient or atmospheric pressure. In the Francis patent the nitrogen gas passes through an exit out of the system. The Savage reference employs pressure to pass the effluent being treated through the filter containing the microorganisms.
Microorganisms have also been used to modify other tobacco components. For example, U.S. Pat. Nos. 4,037,609 and 4,038,993 to Geiss et al. disclose methods for reducing the nicotine content of tobacco by microbial treatment using microorganisms obtained from tobacco, including Pseudomonas putida and Cellulomonas sp. Aerobic fermentation techniques are employed wherein nicotine is degraded via microbial action to 3-succinoylpyridine. The latter microorganism is capable of reducing nitrate to nitrite and actively produces nitrogen gas. Similarly degradation of nicotine to 3-succinoylpyridine by means of the same microorganisms is described in U.S. Pat. No. 4,011,141 to Gravely et al. Lippman et al. U.S. Pat. No. 2,000,855 describes microbial denicotinization of tobacco by fermenting moist tobacco while adding acid to overcome the alkaline condition produced by fermentation. Alternatively the patent suggests removal of volatile bases by supplying an air current or employing suction. Fermentation was used to improve aroma and mellowness in U.S. Pat. No. 2,644,462 to Frankenburg and in U.S. Pat. No. 4,135,521 to Malan et al.
It is generally recognized by those skilled in the art of fermentation that microbial-enzymatic reactions are highly sensitive to forces of a mechanical nature as well as temperature and pH conditions. Carefully controlled, well defined conditions are essential to achieve optimal microbial-enzymatic activity. Utilization of negative pressure during fermentation can affect growth kinetics and production of sequential enzyme systems required during metabolism. Excessive vacuum can result in total destruction of the microorganisms. In fact in food technology, it is widely recognized that the use of a vacuum is effective to contain and control microbial growth and activity during packing and storing of food, particularly meat products. Utilization of a vacuum in microbial processes has thus not been widely used due to this well recognized sensitivity of microorganisms to negative pressures.
Vacuums have been resorted to during alcohol fermentation to counteract alcohol inhibition. For example, U.S. Pat. No. 2,440,925 to Boeckeler, Gerald R. Cysewski et al., "Rapid Ethanol Fermentation Using Vacuum and Cell Recycle," Biotech. and Bioeng. VXIX, pages 1125-1143, (1977) and A. Ramalingam, "Vacuum Alcohol Fermentation," Thesis for Ph.D. at Cornell University (August 1975) all relate to vacuum distillation of alcohol to remove it from the fermentation beer and reduce growth inhibition due to its presence.
Vacuum systems, however, have not been suggested or used to facilitate denitrification processes. Rather, the prior art has suggested the use of atmospheric or positive pressures to enhance the denitrification of various materials containing nitrate-nitrogen. For example, U.S. Pat. 4,039,438 suggests the use of ambient or atmospheric pressures for the denitrification of drain water. U.S. Pat. No. 3,829,377 suggests performing denitrification of waste water under pressurized systems such as from 1 to 10 atmospheres absolute.
We have now unexpectedly discovered that employing a vacuum during denitrification expedites and otherwise improves upon prior processes for reducing the nitrate-nitrogen content of tobacco materials by means of microorganisms. Specifically the vacuum accelerates the fermentation process thereby substantially decreasing the time required for total denitrification. Moreover, we have found that increased amounts of substrate may be treated in the fermentation vessel when a vacuum is used to remove the nitrogen gas as it is formed. Further the end product via the microbial denitration process of the invention is nitrogen gas, which may be withdrawn from the system by the vacuum. The nitrate reduction is thus accompanied by simple removal of the nitrogen from the treated materials. Moreover, the nitrogen gas can be recycled for sparging, thus eliminating troublesome disposal problems experienced in some prior methods of nitrate removal. Furthermore, the process does not require overly expensive equipment and is not energy intensive. In addition, the process is adaptable to all types of tobacco materials, i.e., aqueous tobacco extracts, tobacco leaf, stems, shredded filler, and the like, whereas the known methods are generally applicable to tobacco that is ultimately utilized in reconstituted tobacco sheets.